When a blood vessel is cut or ruptured, hemostasis (prevention of blood loss) is accomplished by several means including vascular spasm, formation of a platelet plug, blood coagulation, and growth of fibrous tissue into the blood clot. The process of hemostasis begins instantaneously after a blood vessel's integrity is compromised when the wall of the vessel contracts, thereby reducing the flow of blood from the ruptured vessel. Platelets then adhere to the end of the blood vessel, and after the platelets agglutinate, fibrin appears at the end of the ruptured vessel. Finally, a fibrin clot forms that stops the flow of blood. Once a blood clot has formed, it becomes invaded by fibroblasts which form connective tissue all throughout the clot.
Many different substances affect blood coagulation or clotting. Unfortunately, many people suffer from defects in the blood clotting mechanism and need to be treated with various pharmaceutical preparations. The most common disorder is a deficiency in functional Factor VIII complex.
Factor VIII complex is well known and is a large molecular weight complex composed of two different proteins, a carrier protein (von Willebrand Factor, vWF) and the procoagulant protein factor (Factor VIII:C). The two proteins are linked together by non-covalent bonds. Von Willebrand factor interacts with platelets in a way that promotes primary hemostasis.
The primary therapeutic use of Factor VIII complex has been its intravenous administration to hemophilia A patients. Hemophilia A is an inherited sex-linked coagulation disorder carried by females and expressed in males. In severe cases, relatively high concentration of Factor VIII complex are required to treat the hemophilia. These high concentrations are usually obtained by purification and concentration of Factor VIII complex from pools of human blood.
A Factor VIII complex product named Alphahate.TM., which is currently available from Alpha Therapeutic Corporation in Los Angeles, Calif., is a highly purified and processed Factor VIII complex that is prepared by means of a heparin affinity column. The method of purification is detailed in U.S. Pat. No. 5,288,853 to Bhattacharya et. al. issued on Feb. 22, 1994, which is hereby incorporated by reference. The Alphanate.TM. product is virus inactivated using methods including organic solvent detergent method. The specific activity of the Alphahate.TM. Factor VIII complex product which contains yon Willebrand factor (vWF) reached more than 100 U/mg of protein. However, using a heparin affinity column has the disadvantage in that the heparin resin has a low binding capacity for Factor VIII complex as well as a low specificity, which leads to low yields.
The technology of monoclonal antibodies has also been utilized for highly purified Factor VIII products, but the disadvantage of this method is the leaking of animal protein to the product. In addition, the cost of monoclonal antibodies is much higher than the conventional chromatography resin.
Currently, there is a need for an inexpensive new purification method using a column that has a high binding capacity for Factor VIII complex and also has a high specificity in order to obtain high yields.